ABSTRACT CRISPR-Cas systems have rapidly advanced from intriguing prokaryotic immune systems to revolutionary tools for biomedical research, antimicrobials, and gene therapy. Advances to-date have consistently relied on a handful of similar, well-characterized CRISPR-Cas systems. In contrast, nature boasts a rich diversity of systems, where the few characterized examples have revealed varying compositions, functions, mechanisms of action, and phylogenetic distributions. These insights raise fundamental questions about why nature would evolve such diverse systems to perform seemingly equivalent functions and what other functions remain to be discovered. Aside from biological insights, answering these questions could also establish a new generation of CRISPR technologies for biomedical research and human therapeutics. The long-term goal of the PI's independent research program is to elucidate the full spectrum of capabilities offered by CRISPR-Cas systems and how these capabilities can be exploited to understand and improve human health. The proposed research program seeks to build on the PI's previous accomplishments in this area through three major avenues: (1) Investigate how CRISPR-Cas systems cope with DNA repair. CRISPR-Cas systems introduce DNA damage that would be undone by endogenous DNA repair pathways?counterproductive for immune defense yet critical for genome editing. Despite this duality, little is known about the natural interplay between endogenous DNA repair pathways and CRISPR function. This research avenue will investigate the propensity of bacterial DNA repair pathways to counteract CRISPR-Cas systems and the extent to which these pathways and systems naturally coexist. (2) Uncover alternative roles of CRISPR-Cas systems. CRISPR-Cas systems are broadly regarded as adaptive immune systems, yet emerging evidence suggests that these systems were naturally co-opted to perform other functions. This avenue will explore the capacity of the highly abundant Type I systems to regulate gene expression and the potential existence of natural examples that perform these functions. Revealing these natural functions has the potential to inform other uses of these widespread systems. (3) Evaluate the versatility and utility of Type I CRISPR-Cas systems. Type I CRISPR-Cas systems represent the most abundant type in nature and offer the ability to degrade DNA in a unidirectional manner. However, little work has been done to understand and exploit these unique capabilities. This avenue will investigate the ability of these systems to exhibit enhanced specificity and to evaluate the performance of a highly compact Type I system when imported into eukaryotic cells. Guiding these research avenues is an overarching hypothesis that CRISPR-Cas systems encompass a rich diversity of functionalities that serve different purposes, where many of these functionalities are waiting to be harnessed for distinct applications in public health.